Current methods of up-regulating the expression of a gene of interest typically require the introduction of extra copies of the gene into a cell, either by using viruses to introduce extra copies of the gene into the host genome or by introducing plasmids that express extra copies of the target gene. By contrast, the present invention provides short activating RNA molecules which can upregulate gene expression, particularly albumin production.
Various studies indicate that short activating RNA (saRNA) can target a promoter region of a gene and activate the gene. Some have proposed a mechanism (Mechanism A) where short double-stranded or single stranded RNAs find a match in a promoter region of a gene to form a complex that binds to the promoter region and removes so-called “off” tags; and another mechanism (Mechanism B) where a cell produces RNA copies of a promoter region that somehow block production of a protein to silence a gene; and where short double-stranded or single stranded RNAs find a match to bind and destroy the RNA copies. These proposed mechanisms (e.g., believed to be one or possibly both) lead to turning “on” a target gene, for example, to provide for production of a protein. FIG. 1 shows approximate graphical diagrams that illustrate Mechanism A 110 (see graphics 112 and 114) and Mechanism B 120 (see graphic 122) with respect to a promoter region, a coding region and a double stranded RNA that can form a complex (see, e.g., Holmes, B., “Turn genes on, turn diseases off”, New Scientist, 7 Apr. 2007, which is incorporated herein by reference).
RNA interference (RNAi) is an important gene regulatory mechanism that causes sequence-specific down-regulation of target mRNAs. RNAi is mediated by “interfering RNA” (iRNA); an umbrella term which encompasses a variety of short double stranded RNA (dsRNA) molecules which function in the RNAi process.
Exogenous dsRNA can be processed by the ribonuclease protein Dicer into double-stranded fragments of 19 to 25 base pairs, preferably 21-23 base pairs, with several unpaired bases on each 3′ end forming a 3′ overhang. Preferably, each 3′ overhang is 1-3, more preferably 2, nucleotides long. These short double-stranded fragments are termed small interfering RNAs (siRNAs) and these molecules effect the down-regulation of the expression of target genes. Since the elucidation of their function, siRNAs have been used as tools to down-regulate specific genes.
A protein complex called the RNA-induced silencing complex (RISC) incorporates one of the siRNA strands and uses this strand as a guide to recognize target mRNAs. Depending on the complementarity between guide RNA and mRNA, RISC then destroys or inhibits translation of the mRNA. Perfect complementarity results in mRNA cleavage and destruction and as result of the cleavage the mRNA can no longer be translated into protein. Partial complementarity—particularly with sites in the mRNA's 3′ untranslated region (UTR)—results in translational inhibition.
Recently it has been discovered that although RISC primarily regulates genes post transcription, RNAi can also modulate gene transcription itself. It is believed that short RNAs regulate transcription by targeting for destruction transcripts that are sense or antisense to the regulated RNA and which are presumed to be non-coding transcripts. Destruction of these non-coding transcripts through RNA targeting has different effects on epigenetic regulatory patterns depending on the nature of the RNA target. Destruction of ncRNA targets which are sense to a given mRNA results in transcriptional repression of that mRNA, whereas destruction of ncRNA targets which are antisense to a given mRNA results in transcriptional activation of that mRNA. By targeting such antisense transcripts, RNAi can therefore be used to up-regulate specific genes.
A published US patent application 2010/0210707 A1 (707 application), which is incorporated by reference herein, sets forth some technology for use of saRNA. The '707 application describes selection of a non-coding region of a nucleic acid sequence of a gene to provide for complementarity of a saRNA strand to, in turn, provide for an increase in transcription of the corresponding gene. Such an approach infers that the “target” is known a priori. Further, the '707 application states explicitly “saRNAs do not target cryptic promoter transcription”. In detail, the '707 application notes that expression of E-cadherin, p21 and GAPDH was detected using gene specific primer sets; that no cryptic transcript was detected using primers complementary to the E-cadherin promoter; and that no cryptic transcript was amplified in the p21 promoter.
The '707 application also describes an saRNA molecule with at least a first ribonucleic acid strand with a 5′ region of complementarity to a non-coding sequence of a gene, where the gene encodes a polypeptide that inhibits cellular proliferation, and a 3′ terminal region of at least one nucleotide non-complementary to the non-coding sequence, where administering of the saRNA provides for an increase in expression of the polypeptide and a decrease in cellular proliferation. As explicitly stated, the referred to polypeptide itself “inhibits cellular proliferation”.
As described herein, various technologies, techniques, etc., can provide for saRNA, optionally without specific a priori knowledge, where such saRNA may be administered, directly or indirectly, to impact cell proliferation. In various examples, cell proliferation is impacted not by presence of a polypeptide that inhibits cell proliferation but rather by a controlling a mechanism (or mechanisms) for production of one or more polypeptides. As described herein, each of such one or more polypeptides may or may not, by presence of the polypeptide molecule itself, inhibit cell proliferation.